RU2011133912A - RESISTANCE OF PLANTS TO OOMYCETES, INFLUENCED BY AN ANTIBODIES Fusion - Google Patents

RESISTANCE OF PLANTS TO OOMYCETES, INFLUENCED BY AN ANTIBODIES Fusion Download PDF

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RU2011133912A
RU2011133912A RU2011133912/10A RU2011133912A RU2011133912A RU 2011133912 A RU2011133912 A RU 2011133912A RU 2011133912/10 A RU2011133912/10 A RU 2011133912/10A RU 2011133912 A RU2011133912 A RU 2011133912A RU 2011133912 A RU2011133912 A RU 2011133912A
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fusion protein
antibody
fragment
plant
phytophthora
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Сильвия ШЛЕКЕР
Дитер ПЕШЕН
Райнер Фишер
Штефан ШИЛЛЬБЕРГ
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ФРАУНХОФЕР-ГЕЗЕЛЛЬШАФТ ЦУР ФЕРДЕРУНГ ДЕР АНГЕВАНДТЕН ФОРШУНГ э.ф.
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Abstract

1. Белок слияния, содержащий по меньшей мере один противооомицетный белок или пептид (ПОБ), связанный с антителом или фрагментом антитела, специфически распознающий эпитоп Oomycota.2. Белок слияния по п.1, где ПОБ выбран из группы, состоящей из Сес, D4E1, GR7, Mag и мечниковина (МТК).3. Белок слияния по п.1 или 2, где антитело или его фрагмент специфически распознает эпитоп Phytophthora ssp., Pythium ssp., Peronospora ssp.и Pseudoperonospora ssp., предпочтительно, Phytophthora infestans, Phytophthora capsici, Phytophthora cactorum, Phytophthora cannamoni или Phytophthora nicotianae.4. Белок слияния по п.1, где антитело или его фрагмент представляет собой F(ab′)2 фрагмент, Fab фрагмент, scFv, биспецифический scFv, триспецифический scFv, диатело, однодоменное антитело (dAb), миниантитело или единицу молекулярного распознавания (ЕМР).5. Белок слияния по п.4, где фрагмент антитела представляет собой scFv, имеющий последовательность, выбранную из группы, состоящей из SEQ ID N0:61 - SEQ ID NO:69.6. Белок слияния по п.1, дополнительно содержащий по меньшей мере одну N-концевую и/или С-концевую метку, по меньшей мере облегчающую обнаружение и/или очистку белка слияния.7. Белок слияния по п.6, где метка выбрана из группы, состоящей из c-myc, his, his, tag54, FLAG, НА, HSV-, T7, S, стрептококковой и Е-метки.8. Белок слияния по п.1, дополнительно содержащий клеточную нацеливающую последовательность.9. Белок слияния по п.8, где клеточная нацеливающая последовательность представляет собой последовательность для выделения или локализации белка слияния в клеточных отделах или органеллах, предпочтительно, в апопласте, вакуоли, внутренних и/или внешних мембранах или просвете эндоплазматического ретикулума (ЭР).10. Белок слияния по п.1, где ПОБ и антитело или его фрагмент связаны пептидным1. A fusion protein containing at least one anti-oomycete protein or peptide (PHB) associated with an antibody or antibody fragment that specifically recognizes the Oomycota epitope. 2. The fusion protein according to claim 1, wherein the PHB is selected from the group consisting of Cec, D4E1, GR7, Mag, and Swordsman (MTK). The fusion protein according to claim 1 or 2, wherein the antibody or fragment thereof specifically recognizes the epitope of Phytophthora ssp., Pythium ssp., Peronospora ssp. And Pseudoperonospora ssp., Preferably Phytophthora infestans, Phytophthora capsici, Phytophthora cactorthnam phththumthophthnamthoma, Phytophthora cactorumthymthum. four. The fusion protein according to claim 1, wherein the antibody or fragment thereof is an F (ab ′) 2 fragment, a Fab fragment, scFv, a bispecific scFv, a trispecific scFv, a diatel, a single domain antibody (dAb), a mini-antibody, or a molecular recognition unit (EMP). 5. The fusion protein of claim 4, wherein the antibody fragment is scFv having a sequence selected from the group consisting of SEQ ID N0: 61 - SEQ ID NO: 69.6. The fusion protein of claim 1, further comprising at least one N-terminal and / or C-terminal tag, at least facilitating the detection and / or purification of the fusion protein. The fusion protein according to claim 6, wherein the label is selected from the group consisting of c-myc, his, his, tag54, FLAG, HA, HSV-, T7, S, streptococcal and E-labels. The fusion protein of claim 1, further comprising a cell targeting sequence. The fusion protein of claim 8, wherein the cell targeting sequence is a sequence for isolating or localizing the fusion protein in cell divisions or organelles, preferably in the apoplast, vacuole, internal and / or external membranes or lumen of the endoplasmic reticulum (ER). 10. The fusion protein of claim 1, wherein the PHB and the antibody or fragment thereof are linked by a peptide

Claims (22)

1. Белок слияния, содержащий по меньшей мере один противооомицетный белок или пептид (ПОБ), связанный с антителом или фрагментом антитела, специфически распознающий эпитоп Oomycota.1. A fusion protein containing at least one anti-oomycete protein or peptide (PHB) associated with an antibody or antibody fragment that specifically recognizes an Oomycota epitope. 2. Белок слияния по п.1, где ПОБ выбран из группы, состоящей из Сес, D4E1, GR7, Mag и мечниковина (МТК).2. The fusion protein according to claim 1, wherein the PHB is selected from the group consisting of Cec, D4E1, GR7, Mag, and Swordsman (MTK). 3. Белок слияния по п.1 или 2, где антитело или его фрагмент специфически распознает эпитоп Phytophthora ssp., Pythium ssp., Peronospora ssp.и Pseudoperonospora ssp., предпочтительно, Phytophthora infestans, Phytophthora capsici, Phytophthora cactorum, Phytophthora cannamoni или Phytophthora nicotianae.3. The fusion protein according to claim 1 or 2, wherein the antibody or fragment thereof specifically recognizes the epitope of Phytophthora ssp., Pythium ssp., Peronospora ssp. And Pseudoperonospora ssp., Preferably Phytophthora infestans, Phytophthora capsici, Phytophthora cacththum, Phytophthora cacththum, nicotianae. 4. Белок слияния по п.1, где антитело или его фрагмент представляет собой F(ab′)2 фрагмент, Fab фрагмент, scFv, биспецифический scFv, триспецифический scFv, диатело, однодоменное антитело (dAb), миниантитело или единицу молекулярного распознавания (ЕМР).4. The fusion protein according to claim 1, where the antibody or its fragment is an F (ab ′) 2 fragment, Fab fragment, scFv, bispecific scFv, trispecific scFv, diabody, single domain antibody (dAb), miniantibody or molecular recognition unit (EMP ) 5. Белок слияния по п.4, где фрагмент антитела представляет собой scFv, имеющий последовательность, выбранную из группы, состоящей из SEQ ID N0:61 - SEQ ID NO:69.5. The fusion protein according to claim 4, where the antibody fragment is an scFv having a sequence selected from the group consisting of SEQ ID N0: 61 - SEQ ID NO: 69. 6. Белок слияния по п.1, дополнительно содержащий по меньшей мере одну N-концевую и/или С-концевую метку, по меньшей мере облегчающую обнаружение и/или очистку белка слияния.6. The fusion protein according to claim 1, further comprising at least one N-terminal and / or C-terminal tag, at least facilitating the detection and / or purification of the fusion protein. 7. Белок слияния по п.6, где метка выбрана из группы, состоящей из c-myc, his6, his5, tag54, FLAG, НА, HSV-, T7, S, стрептококковой и Е-метки.7. The fusion protein according to claim 6, where the label is selected from the group consisting of c-myc, his 6 , his 5 , tag54, FLAG, HA, HSV-, T7, S, streptococcal and E-tags. 8. Белок слияния по п.1, дополнительно содержащий клеточную нацеливающую последовательность.8. The fusion protein of claim 1, further comprising a cell targeting sequence. 9. Белок слияния по п.8, где клеточная нацеливающая последовательность представляет собой последовательность для выделения или локализации белка слияния в клеточных отделах или органеллах, предпочтительно, в апопласте, вакуоли, внутренних и/или внешних мембранах или просвете эндоплазматического ретикулума (ЭР).9. The fusion protein of claim 8, wherein the cell targeting sequence is a sequence for isolating or localizing the fusion protein in cell divisions or organelles, preferably in the apoplast, vacuole, internal and / or external membranes or lumen of the endoplasmic reticulum (ER). 10. Белок слияния по п.1, где ПОБ и антитело или его фрагмент связаны пептидным линкером.10. The fusion protein according to claim 1, where the PHB and the antibody or its fragment are linked by a peptide linker. 11. Белок слияния по п.10, содержащий последовательность, выбранную из группы, состоящей из SEQ ID NO:76 - SEQ ID NO: 120.11. The fusion protein of claim 10, containing a sequence selected from the group consisting of SEQ ID NO: 76 - SEQ ID NO: 120. 12. Полинуклеотид, содержащий последовательность, кодирующую белок слияния по любому из пп.1-11.12. Polynucleotide containing the sequence encoding a fusion protein according to any one of claims 1 to 11. 13. Полинуклеотид по п.12, где последовательность оптимизирована для экспрессии в клетке хозяина.13. The polynucleotide of claim 12, wherein the sequence is optimized for expression in a host cell. 14. Вектор, содержащий полинуклеотид по п.12 или 13 в составе экспрессионной кассеты.14. A vector containing the polynucleotide of claim 12 or 13 as part of an expression cassette. 15. Вектор по п.14, где экспрессионная кассета функционально связана с одной или более чем одной регуляторной последовательностью, позволяя экспрессию белка слияния, предпочтительно в растениях, органах растений, растительных тканях и/или растительных клетках.15. The vector of claim 14, wherein the expression cassette is operably linked to one or more than one regulatory sequence, allowing expression of the fusion protein, preferably in plants, plant organs, plant tissues and / or plant cells. 16. Клетка хозяина, содержащая полинуклеотид по п.12 или 13 и/или вектор по п.14 или 15.16. A host cell containing a polynucleotide according to claim 12 or 13 and / or a vector according to 14 or 15. 17. Способ получения белка слияния по любому из пп.1-11, при котором осуществляют стадии:17. A method of producing a fusion protein according to any one of claims 1 to 11, wherein the steps are: (а) культивирование клеток хозяина по п.16 в культуральной среде в условиях, позволяющих экспрессию белка слияния по любому из пп.1-11; и(a) culturing the host cells according to clause 16 in a culture medium under conditions allowing expression of the fusion protein according to any one of claims 1 to 11; and (б) выделение белка слияния из среды и/или клеток хозяина.(b) isolation of the fusion protein from the medium and / or host cells. 18. Способ получения растения, устойчивого к Oomycota, растительной клетки или растительной ткани, содержащий стадию введения полинуклеотида по п.12 или 13 в геном растения, растительной клетки или растительной ткани.18. A method for producing a plant resistant to Oomycota, a plant cell or plant tissue, comprising the step of introducing a polynucleotide according to claim 12 or 13 into the genome of a plant, plant cell or plant tissue. 19. Трансгенное растение или растительная ткань, трансформированная полинуклеотидом по п.12 или 13.19. A transgenic plant or plant tissue transformed with a polynucleotide according to claim 12 or 13. 20. Собираемые части и материалы для распространения, полученные из трансгенного растения по п.19.20. Collected parts and materials for distribution obtained from a transgenic plant according to claim 19. 21. Применение белка слияния по любому из пп.1-11, полинуклеотида по п.12 или 13 и/или вектора по п.14 или 15 для защиты растения от действия Oomycota.21. The use of the fusion protein according to any one of claims 1 to 11, the polynucleotide according to claim 12 or 13, and / or the vector according to claim 14 or 15, for protecting the plant from the action of Oomycota. 22. Набор, содержащий белок слияния по любому из пп.1-11, и/или полинуклеотид по п.12 или 13, и/или вектор по п.14 или 15, и/или клетку хозяина по п.16 вместе со средствами обнаружения указанного белка слияния, полинуклеотида, вектора и/или клетки хозяина. 22. A kit containing a fusion protein according to any one of claims 1 to 11, and / or a polynucleotide according to claim 12 or 13, and / or a vector according to claim 14 or 15, and / or a host cell according to claim 16, together with means detecting said fusion protein, polynucleotide, vector and / or host cell.
RU2011133912/10A 2009-08-24 2010-08-05 RESISTANCE OF PLANTS TO OOMYCETES, INFLUENCED BY AN ANTIBODIES Fusion RU2011133912A (en)

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